Bmp gene and fusion protein

ABSTRACT

This invention relates to BMP fusion genes, BMP fusion proteins, and methods for making BMP fusion genes and BMP fusion proteins. The invention further relates to methods for treatment using BMP fusion genes and BMP fusion proteins. Additionally, the invention relates to BMP fusion gene and BMP fusion protein pharmaceutical compositions.

This application is a 371 National Phase of International ApplicationNo. PCT/US2005/038885, filed Oct. 26, 2005, which claims priority toU.S. provisional patent Application No. 60/622,490, filed Oct. 27, 2004.The contents of these two applications are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to a gene encoding a bone morphogenetic proteinfusion protein (“BMP fusion gene”), a BMP fusion protein, methods forproducing a BMP fusion protein, and methods for treatment using a BMPfusion gene or a BMP fusion protein.

BACKGROUND

Bone morphogenetic proteins (BMPs) are proteins, which induce boneformation. BMPs are members of the transforming growth factor beta(TGF-β) superfamily of dimeric, disulfide-linked growth factors(Sampath, et al., J Biol. Chem. 1990; 265:13198-13205). BMP2 and BMP7(also known as osteogenic protein-1) were initially co-purified frombovine bone. Two or more BMP genes are often co-expressed, for example,co-localization of BMP2 and BMP7 transcripts have been demonstrated indeveloping limbs of mouse embryos (Lyons et al., Mech Dev. 1995;50:71-83). Other studies of embryogenesis have also shown theco-expression of several pairs of BMP genes is required for normal bonedevelopment (Katagiri et al., Dev Genet. 1998; 22:340-348; Solloway etal., Development. 1999; 126:1753-1768). In vitro studies suggest thatco-expression of BMPs can result in the expression of heterodimeric BMPs(Aono et al., Biochem Biophys Res Commun. 1995; 210:670-677; Hazama etal., Biochem Biophys Res Commun. 1995; 209:859-866; Israel et al.,Growth Factors. 1996; 13:291-300). However, “native” BMP heterodimershave not been isolated in vivo. Osteoblastic differentiation and ectopicbone formation studies have shown that BMP heterodimers are more potentthan their respective homodimers (Aono et al.; Hazama et al.; Israel, etal.).

Spine fusion surgery is a commonly performed orthopedic procedure, whichrequires the formation of new bone around the spine to increase itsstability. Non-union, or the failure of new bone to form, is a majorcomplication of spine fusion, which occurs in up to 26% ofposterolateral lumbar spine fusion cases (Steinman et al., Clin Ortho.1992; 284:80-90; Rawlings et al., Spine. 1994; 8:563-571; Kimura et al.,J Spinal Disord. 2001; 14:301-310). Autogenous bone grafting is the goldstandard for induction of a spine fusion, but harvesting of the bonegraft (typically from the iliac crest) is associated with significantmorbidity in up to 30% of patients (Steinman et al.; Rawlings et al.;Kimura et al.; Arrington et al., Clin Ortho. 1996; 329:300-309). Assuch, the development of graft alternatives such as BMPs is of greatinterest. Recombinant BMP (rhBMP) homodimers have been shown to enhancespine fusion in animals and humans, but the doses required are extremelyhigh (Celeste et al., Proc Ntl Aca Sci USA. 1990; 87:9843-9847; Boden etal., Spine. 2002; 27:2662-2673; Cook S D, Orthopedics. 1999; 22:669-671;Friedlander G E, J Bone Joint Surg Am. 2001; 83-A Suppl 1(Pt2):S160-161; Sandhu et al., Spine. 2002; 27(16 Suppl 1):S32-38; Schmittet al., J Ortho Res. 1999; 17:269-278; Wozney J M, Spine. 2002; 27(16Suppl 1):S2-8; Zlotolow et al., J Am Acad Orth Surg. 2000; 8:3-9).Recombinant BMP2 (InFUSE, available from Medtronic) is supplied as apowder, which is mixed with sterile water and applied to an absorbablecollagen sponge (available from Integra) prior to its topicalapplication (i.e., direct application to the bone or to the vicinity ofthe bone). Recombinant BMP7 has been used in combination with a collagencarrier (the combination is marketed as OP-1 Implant, available fromStryker) to induce bone formation. OP-1 Implant is wetted to form apaste that is surgically implanted in a bone fracture gap.

Currently available rhBMPs are homodimers with two identical monomerslinked by a disulfide bond. Post-translational processing of homodimerBMP proteins requires dimer formation followed by cleavage of thepro-proteins. Relative to homodimers, heterodimeric BMPs are more potentinducers of osteoblastic differentiation in vitro and enhancers of boneformation in vivo. BMP heterodimers are produced by co-transfection oftarget cells with two different BMP genes, which results in theproduction of both heterodimers and a mixture of homodimers. Because themonomers BMP2 and BMP7 are similar, BMP 2/7 heterodimers are difficultto purify from BMP2 homodimers, BMP7 homodimers, or a mixture thereof(Wozney J M. Mol Repro Devel. 1992; 32:160-167; Celeste et al., ProcNatl Acad Sci USA 1990; 87:9843-9847.

SUMMARY OF THE INVENTION

The present invention provides a gene encoding two different BMPproteins (“BMP fusion gene”) in tandem, which results in expression of aBMP fusion protein (e.g., a BMP-2/7 fusion protein). A BMP fusion geneaccording to the present invention results in the expression of a singlechain polypeptide, which contains both “halves” of a BMP heterodimer andforms by folding rather than dimerization. The BMP fusion gene and theBMP fusion protein of the present invention provide a BMP fusion proteinequipotent to heterodimeric BMP. A BMP fusion gene of the presentinvention comprises a first BMP gene, a linker, and a second, differentBMP gene, wherein the linker replaces the first BMP gene stop codon; thesecond, different BMP start codon; and the second, different BMP signalpeptide nucleotide sequence. A preferred linker is comprised of about 60base pairs (“bp”). An especially preferred linker encodes the amino acidsequence (Gly₄Ser)₄. A preferred BMP fusion gene is a human BMP fusiongene.

The present invention also provides a BMP fusion gene encoding a BMPprotein component, a linker, and a nucleotide sequence encoding a TGF-βsuperfamily protein component, wherein the TGF-β superfamily proteincomponent is different than the BMP protein component. Further, theinvention provides a BMP fusion gene encoding a BMP-7/GDF-7;BMP-15/GDF-9; BMP-2/TGF-β1 or BMP-4/TGF-β1 fusion protein. An embodimentof the present invention provides a gene encoding BMP2 and BMP7 intandem, which results in expression of a BMP2/7 fusion protein (i.e., a“BMP-2/7 fusion gene”). A BMP-2/7 fusion gene according to the presentinvention results in the expression of a single chain polypeptide, whichcontains both “halves” of a BMP-2/7 heterodimer and forms by foldingrather than dimerization. The BMP-2/7 fusion gene and the BMP-2/7 fusionprotein of the present invention provide a BMP-2/7 fusion proteinequipotent to heterodimeric BMP-2/7. A BMP-2/7 fusion gene of thepresent invention comprises a BMP2 gene, a linker, and a BMP7 gene,wherein the linker replaces the BMP2 stop codon, the BMP7 start codon,and the BMP7 signal peptide nucleotide sequence. A preferred linker iscomprised of about 60 base pairs (“bp”). An especially preferred linkerencodes the amino acid sequence (Gly₄Ser)₄. A preferred BMP-2/7 fusiongene is a human BMP-2/7 fusion gene.

In an aspect of the present invention, a BMP fusion protein includes afirst BMP protein component, a linker, and a second, different BMPprotein component. A preferred BMP fusion protein is a human BMP fusionprotein. A preferred linker is comprised of about 20 amino acids. Anespecially preferred linker is the amino acid sequence (Gly₄Ser)₄.

In another aspect of the present invention, a BMP fusion proteinincludes a BMP protein component, a linker, and a nucleotide sequenceencoding a TGF-β superfamily protein component, wherein the TGF-βsuperfamily protein component is different than the BMP proteincomponent. Further, according to the present invention, a BMP fusionprotein is a BMP-7/GDF-7; BMP-15/GDF-9; BMP-2/TGF-β1 or BMP-4/TGF-β1fusion protein.

According to the present invention, a BMP fusion protein comprises:

(a) a first BMP amino acid sequence as set forth in any one of SEQ IDNOS:2, 4 or 10 to 64;

(b) a linker as set forth in SEQ ID NO:5; and

(c) a second, different BMP amino acid sequence as set forth in any oneof SEQ ID NOS:2, 4 or 10 to 64;

wherein the BMP amino acid sequence of (a) is different than the BMPamino acid sequence of (b) and either (a) or (b) is a BMP amino acidsequence as set forth in any one of SEQ ID NOs:2, 4 or 10 to 39.

Recombinant nucleic acids according to the present invention provide forefficient expression of BMP fusion gene constructs. Also encompassed areexpression vectors in which the BMP fusion gene is operably associatedwith an expression control sequence. The invention extends to host cellstransfected or transformed with the BMP fusion gene expression vector.The BMP fusion protein can be produced by isolating it from host cellsgrown under conditions that permit expression of the construct.

The methods of making a BMP fusion protein according to the presentinvention provide significant advantages over known methods ofheterodimeric BMP production because a preparation is produced free ofBMP homodimers, thus avoiding difficult, time-consuming and expensiveseparation of BMP heterodimers from BMP homodimers. Moreover, because ofits increased potency, a BMP fusion protein can be administered in lowerdoses relative to BMP homodimers.

In one aspect, the present invention provides a method for producing arecombinant BMP fusion protein having bone stimulating activitycomprising culturing a host cell containing a nucleotide sequenceencoding BMP gene, and isolating the biologically active fusion proteinfrom the culture medium.

Further, according to methods of the present invention, the BMP fusiongene or the BMP fusion protein can be administered to a patient toinduce local or systemic bone formation.

A BMP-2/7 fusion protein of the present invention comprises a BMP2protein component, a linker, and a BMP7 protein component. A preferredBMP-2/7 fusion protein is a human BMP-2/7 fusion protein. A preferredlinker is comprised of about 20 amino acids. An especially preferredlinker is the amino acid sequence (Gly₄Ser)₄.

In another aspect of the present invention, a BMP-2/7 fusion proteincomprises:

(a) a BMP2 amino acid sequence as set forth in SEQ ID NO:2;

(b) a linker as set forth in SEQ ID NO:5; and

(c) a BMP7 amino acid sequence as set forth in SEQ ID NO:4.

Recombinant nucleic acids according to the present invention provide forefficient expression of BMP-2/7 fusion gene constructs. Also encompassedare expression vectors in which the BMP-2/7 fusion gene is operablyassociated with an expression control sequence. The invention extends tohost cells transfected or transformed with the BMP-2/7 gene expressionvector. The BMP-2/7 fusion protein can be produced by isolating it fromthe host cells grown under conditions that permit expression of theconstruct.

The methods of making a BMP-2/7 fusion protein according to the presentinvention provide significant advantages over known methods ofheterodimeric BMP-2/7 production because a preparation is produced freeof BMP homodimers, thus avoiding difficult, time-consuming and expensiveseparation of BMP heterodimers from BMP homodimers. Moreover, because ofits increased potency, a BMP-2/7 fusion protein can be administered inlower doses relative to BMP homodimers.

In one aspect, the present invention provides a method for producing arecombinant BMP-2/7 fusion protein having bone stimulating activitycomprising culturing a host cell containing a nucleotide sequenceencoding BMP-2/7 gene, and isolating the biologically active fusionprotein from the culture medium.

Further, according to methods of the present invention, the BMP-2/7fusion gene or the BMP-2/7 fusion protein can be administered to apatient to induce local or systemic bone formation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the mRNA sequence (SEQ ID NO:1) (Genbank Accession #M22489)and amino acid sequence (SEQ ID NO: 2) of human BMP2. The stop codon ofBMP2 is underlined and bold. Forward and reverse PCR primer sequencesare shaded.

FIG. 2 shows the mRNA sequence (SEQ ID NO: 3) (Genbank Accession#X51801) and amino acid sequence (SEQ ID NO: 4) of human BMP7. The startcodon and signal peptide nucleotide sequence of BMP7 are underlined andbold. Forward and reverse PCR primer sequences are shaded.

FIG. 3 illustrates construction of a BMP-2/7 fusion gene using serialPCR reactions.

FIG. 4 depicts a graph, which shows BMP7 content in supernatants ofcells transfected with pShuttleCMV-BMP-2/7, pCMV-GFP or medium onlyfollowing immunoprecipitation with anti-BMP2 antibody.

FIG. 5 depicts graphs, which show OCN expression in C2C12 cellsstimulated by A549 cell supernatants containing BMP-217 fusion protein(FIG. 5 a) or BMP-2/7 heterodimer generated by co-transfection with BMP2and BMP7 genes (FIG. 5 b). FIG. 5 a also shows that BMP-2/7 fusionprotein at 2 ng/ml (1:5 dilution) resulted in an OCN level comparable to1000 ng/ml of rhBMP2 or rhBMP7.

FIG. 6 depicts a graph, which shows OCN levels induced by maximal doses(i.e., about 1000 ng/ml) of rhBMP2 or rhBMP7.

FIG. 7 shows the amino acid sequence of human BMP3 precursor (SEQ IDNO:10, Genbank Accession #NP_(—)001192) and human BMP3A precursor (SEQID NO:11, Genbank Accession #P12645).

FIG. 8 shows the amino acid sequence of human BMP3B precursor (SEQ IDNO:12, Genbank Accession #P55107) and human BMP3B (SEQ ID NO:13, GenbankAccession #BAA08453).

FIG. 9 shows the amino acid sequence of human BMP3B (SEQ ID NO:14,Genbank Accession #BAA008452, and SEQ ID NO:15, Genbank Accession#NP_(—)004953).

FIG. 10 shows the amino acid sequence of human BMP4 precursor (SEQ IDNO:16, Genbank Accession #P12644) and human BMP4 preprotein (SEQ IDNO:17, Genbank Accession #NP_(—)001193).

FIG. 11 shows the amino acid sequence of human BMP4 preprotein (SEQ IDNO:18, Genbank Accession #NP_(—)570911, and SEQ ID NO:19, GenbankAccession #NP_(—)570912).

FIG. 12 shows the amino acid sequence of human BMP4 (SEQ ID NO:20,Genbank Accession #BAA06410, and SEQ ID NO:21, Genbank. Accession#AAC72278).

FIG. 13 shows the amino acid sequence of human BMP5 preprotein (SEQ IDNO:22, Genbank Accession #NP_(—)066551) and human BMP5 precursor (SEQ IDNO:23, Genbank Accession #P22003).

FIG. 14 shows the amino acid sequence of human BMP6 precursor (SEQ IDNO:24, Genbank Accession #P22004, and SEQ ID NO:25, Genbank Accession#NP_(—)001709).

FIG. 15 shows the amino acid sequence of human BMP8B preprotein (SEQ IDNO:26, Genbank Accession #NP_(—)001711) and human BMP8B (SEQ ID NO:27,Genbank Accession #P34820).

FIG. 16 shows the amino acid sequence of human BMP9 (SEQ ID NO:28,Genbank Accession #Q9UK05, and SEQ ID NO:29, Genbank Accession#NP_(—)057288).

FIG. 17 shows the amino acid sequence of human BMP10 preprotein (SEQ IDNO:30, Genbank Accession #NP_(—)055297) and human BMP10 precursor (SEQID NO:31, Genbank Accession #O95393).

FIG. 18 shows the amino acid sequence of human BMP10 (SEQ ID NO:32,Genbank Accession #AAC77462) and human BMP11 (SEQ ID NO:33, GenbankAccession #AAC72852).

FIG. 19 shows the amino acid sequence of human BMP11 (SEQ ID NO:34,Genbank Accession #NP_(—)005802, and SEQ ID NO:35, Genbank Accession#O95390).

FIG. 20 shows the amino acid sequence of human BMP15 precursor (SEQ IDNO:36, Genbank Accession #O95972, and SEQ ID NO:37, Genbank Accession#NP_(—)005439).

FIG. 21 shows the amino acid sequence of human TGFβ BMP (SEQ ID NO:38,Genbank Accession #AAA36737) and human BMPY (SEQ ID NO:39, Genbank.Accession #AAF15295).

FIG. 22 shows the amino acid sequence of human embryonic GDF1 precursor(SEQ ID NO:40, Genbank Accession #P27539) and human GDF1 (SEQ ID NO:41,Genbank Accession #NP_(—)001483).

FIG. 23 shows the amino acid sequence of human GDF3 precursor (SEQ IDNO:42, Genbank Accession #NP_(—)065685 and SEQ ID NO:43, GenbankAccession #Q9NR23).

FIG. 24 shows the amino acid sequence of human GDF5 precursor (SEQ IDNO:44, Genbank Accession #P43026) and human GDF5n preprotein (SEQ IDNO:45, Genbank Accession #NP_(—)000548).

FIG. 25 shows the amino acid sequence of bovine GDF6 precursor (SEQ IDNO:46, Genbank Accession #P55106) and human GDF8 precursor (SEQ IDNO:47, Genbank Accession #O14793).

FIG. 26 shows the amino acid sequence of human GDF8 (SEQ ID NO:48,Genbank Accession #NP_(—)005250) and human GDF9 precursor (SEQ ID NO:49,Genbank Accession #NP_(—)005251).

FIG. 27 shows the amino acid sequence of human GDF10 (SEQ ID NO:50,Genbank Accession #AAH28237) and human GDF15 precursor (SEQ ID NO:51,Genbank Accession #Q99988).

FIG. 28 shows the amino acid sequence of human GDF15 (SEQ ID NO:52,Genbank Accession #NP_(—)004855) and TGFβ (SEQ ID NO:53, GenbankAccession #AAA36738).

FIG. 29 shows the amino acid sequence of human TGFβ1 (SEQ ID NO:54,GenBank Accession #AAL27646, and SEQ ID NO:55, GenBank Accession#NP_(—)000651).

FIG. 30 shows the amino acid sequence of human TGFβ2 precursor (SEQ IDNO: 56, GenBank Accession #P61812, and SEQ ID NO:57, GenBank Accession#AAA50404).

FIG. 31 shows the amino acid sequence for human TGFβ2 (SEQ ID NO:58,GenBank Accession #AAA50405, and SEQ ID NO:59, GenBank Accession#NP_(—)003229).

FIG. 32 shows the amino acid sequence of human TGFβ3 precursor (SEQ IDNO:60, GenBank Accession #P10600) and human TGFβ3 (SEQ ID NO:61, GenBankAccession #AAH18503).

FIG. 33 shows the amino acid sequence of human TGFβ3 (SEQ ID NO:62,GenBank Accession #CAA33024, and SEQ ID NO:63, GenBank Accession#AAC79727).

FIG. 34 shows the amino acid sequence of human TGFβ3 (SEQ ID NO:64,GenBank Accession #NP_(—)003230).

FIG. 35 shows the results of a reverse transcriptase PCR (RT-PCR) twodays after transfection of A549 epithelial cells. A 2.56 kb band, theexpected size for a BMP 2/7 fusion gene, was detected in the cellstransfected with pSCMV-BMP 2/7 (Lane 2), but not in cells transfectedwith pCMV-GFP (Lane 3) or medium-only control (Lane 4). Neither BMP2cDNA transcripts alone (expected size 1.2 kb) nor BMP7 cDNA transcriptsalone (expected size 1.4 kb) were detected in the pSCMV-BMP2/7transfected cells.

FIG. 36 shows the results of Western blotting using anti-BMP2 antibody.The majority of mature BMP peptides in supernatants from cellstransfected with pSCMV-BMP-2/7 migrated as an immunoreactive band atapproximately 39 kDa under non-reducing conditions. This is the expectedsize for a peptide composed of BMP2, linker and BMP7 (Lane 1). Underreducing conditions, some mature peptides migrated further as productswith molecular masses between approximately 15 to approximately 18 kDa(Lane 1A). A similar pattern of migration of mature BMP peptides (atapproximately 39 kDa and between approximately 15 kDa to approximately18 kDa) was detected by anti-BMP2 antibody in supernatants from cellstransfected with pSCMV-BMP2/7 that had been immunoprecipitated witheither anti-BMP7 antibody (Lane 4) or anti-BMP2 antibody (Lane 6) priorto Western blotting. The “flow-through” portion of the samples collectedfrom the immunoprecipitation columns did not show bands reacting withanti-BMP-2 antibody (Lanes 7-10). A broad band between 45 kDa to 55 kDawas detected in cells transfected with pSCMV-BMP2/7 (Lanes 4 and 6).Western blotting using anti-BMP7 antibody yielded similar results (datanot shown).

FIG. 37 is a bar graph which shows that the levels of BMP2 and BMP7measured by ELISA were similar in the supernatant of cells transfectedwith pSCMV-BMP2/7. Control supernatants did not contain detectable BMPlevels.

FIG. 38 is a bar graph which shows that supernatants of cellstransfected with pSCMV-BMP2/7 contained BMP7 as measured by ELISA afterimmunoprecipitation with anti-BMP2 antibody. Controls did not containdetectable BMP7.

DETAILED DESCRIPTION

Autologous bone grafting is typically used to provide bone to areas ofbone loss in a patient. Such bone loss can occur, for example, as partof a planned orthopedic procedure (e.g., spine fusion surgery) or as aresult of trauma (e.g., fractures with avulsed or missing bone).Autologous bone grafting is associated with a high rate of non-union orfailure of bone formation (e.g., up to 26% of spine fusion cases) andsignificant pain at the donor site (usually the iliac crest). Thus,there exists a need for compositions and methods, which result inefficient induction of bone formation in a patient and reduce the painassociated with autologous bone grafting.

BMP homodimers have been used to replace or supplement autologous bonegrafting. BMP homodimer treatment has met with some success, but highdoses are required and BMP homodimers (e.g., a dimer composed of twoBMP2 monomers) are not as potent as BMP heterodimers (e.g., a dimercomposed of a BMP2 monomer and a BMP7 monomer). BMP heterodimertreatment is limited by the time, labor and cost consuming processrequired to produce the heterodimers. Heterodimers are produced by hostcells co-transfected with nucleic acids encoding each monomer. Themonomers are expressed by the host cells and can dimerize into one oftwo homodimers (e.g., BMP2 homodimer or a BMP7 homodimer) or aheterodimer (e.g., a BMP-2/7 heterodimer). Because of the similaritybetween BMP monomers, separation of the heterodimers from the homodimersis difficult.

BMP fusion genes and BMP fusion proteins of the present invention resultin more effective treatment for bone loss than BMP monomers because BMPfusion protein is as potent as BMP heterodimer. Expression of the BMPfusion gene results in one polypeptide, which folds to include afunctional first BMP protein component and a functional second,different BMP protein component. Because there is no dimerization and noBMP homodimers are formed, BMP fusion protein production avoids theco-transfection and separation steps required for BMP heterodimerproduction. The present invention also provides methods for treatment ofpatients with bone loss by administering a BMP fusion protein or BMPfusion gene whereby autologous bone grafting may be replaced orsupplemented to provide a higher success rate and, possibly, lesspatient pain.

DEFINITIONS

A “BMP fusion gene” as used herein means a nucleotide sequence encodinga BMP fusion protein (e.g., a BMP 2/7 fusion protein). A “human BMPfusion gene” means a BMP fusion gene wherein the nucleotide sequenceencoding the first BMP protein component and the nucleotide sequenceencoding the second, different BMP protein component are human BMPnucleotide sequences. Preferably, the linker nucleotide sequence isfound in the human genome. A “BMP fusion gene” as used hereinencompasses a nucleotide sequence encoding a BMP fusion protein, whichincludes a BMP protein component and a TGF-β superfamily proteincomponent, wherein the TGF-β superfamily protein component is adifferent protein than the BMP protein component (e.g., a BMP-15/GDF-9fusion protein).

A “BMP fusion protein” as used herein means a protein, which includes afirst BMP protein component, a linker, and a second, different BMPprotein component. A “human BMP fusion protein” means a BMP fusionprotein wherein the first BMP protein component and the second,different BMP protein component are human BMP protein components. Asused herein “BMP fusion protein” encompasses a protein, which includes aBMP protein component, a linker, and a TGF-β superfamily proteincomponent, wherein the TGF-β superfamily protein component is adifferent protein component than the BMP protein component (e.g.,BMP-15/GDF-9 fusion protein).

A “linker” as used herein is (1) a nucleotide sequence within a geneencoding a BMP fusion protein, which encodes an amino acid sequence, andbridges a nucleotide sequence encoding the first BMP protein componentand a nucleotide sequence encoding a second, different BMP proteincomponent of the BMP fusion protein, or (2) an amino acid sequence,which bridges the first BMP protein component and the second, differentBMP protein component of a BMP fusion protein. A linker according to thepresent invention can be any nucleotide sequence encoding an amino acidlong enough and flexible enough to permit protein folding and not solong as to introduce additional or erroneous folds, extraneous secondaryor tertiary folds or other errors to the protein structure. A linker ispreferably about 60 bp (about 20 amino acids). An especially preferredlinker is (Gly₄Ser)₄ (SEQ ID NO:5).

“Peptidomimetic” as used herein refers to a compound in which at least aportion of the BMP fusion protein is modified, such that the threedimensional structure of the peptidomimetic remains substantially thesame as that of the functional BMP protein components of the BMP fusionprotein. Alternatively, at least a portion of the BMP fusion protein maybe replaced with a nonpeptide structure, such that the three-dimensionalstructure of the functional BMP protein components of the BMP fusionprotein is substantially retained. In addition, other peptide portionsof the BMP fusion protein may, but need not, be replaced with anonpeptide structure. A variety of peptide modifications are known inthe art and can be used to generate peptidomimetic compounds. See, forexample, International Publication No. WO 01/53331, the contents ofwhich are hereby incorporated in their entirety.

“Subject” or “patient” as used herein means an animal, preferably amammal, and more preferably a human. Typically a subject or patient isin need of bone formation due to, for example, surgical loss of bone,loss of bone due to traumatic injury, or congenitally missing bone.Additionally, a subject or patient can be any animal, including alaboratory animal in the context of a clinical trial or screening oractivity experiment.

The term “therapeutically effective amount” is used herein to mean anamount or dose of a BMP fusion gene or a BMP fusion protein sufficientto induce bone growth in a patient. Alternatively, a therapeuticallyeffective amount of a BMP fusion gene or BMP fusion protein is an amountsufficient to supplement bone formation by known compositions andmethods in the art (e.g., autologous bone grafting).

The term Aabout@ or Aapproximately@ means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system or thedegree of precision required for a particular purpose. For example,Aabout@ can mean within 1 or more than 1 standard deviations, per thepractice in the art. Alternatively, Aabout@ can mean a range of up to20%, preferably up to 10%, more preferably up to 5%, and more preferablystill up to 1% of a given value.

Molecular Biology

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

“Amplification” of DNA as used herein denotes the use of polymerasechain reaction (PCR) to increase the concentration of a particular DNAsequence within a mixture of DNA sequences. For a description of PCR seeSaiki et al, Science 1988, 239:487.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear (e.g., restrictionfragments) or circular DNA molecules, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in a nucleic acid, such as DNA andRNA, and means any chain of two or more nucleotides. A nucleotidesequence typically carries genetic information, including theinformation used by cellular machinery to make proteins and enzymes.These terms include double or single stranded genomic and cDNA, RNA, anysynthetic and genetically manipulated polynucleotide, and both sense andanti-sense polynucleotide (although only sense stands are beingrepresented herein). This includes single- and double-strandedmolecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as“protein nucleic acids” (PNA) formed by conjugating bases to an aminoacid backbone. This also includes nucleic acids containing modifiedbases, for example thio-uracil, thio-guanine and fluoro-uracil.

The nucleic acids herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. The promoter may be operatively associated with otherexpression control sequences, including enhancer and repressorsequences.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

A coding sequence is “under the control of” or “operatively associatedwith” expression control sequences in a cell when RNA polymerasetranscribes the coding sequence into RNA, particularly mRNA, which isthen trans-RNA spliced (if it contains introns) and translated into theprotein encoded by the coding sequence.

The term “expression control sequence” refers to a promoter and anyenhancer or suppression elements that combine to regulate thetranscription of a coding sequence. In a preferred embodiment, theelement is an origin of replication.

The terms “vector”, “cloning vector” and “expression vector” refer tothe vehicle by which DNA can be introduced into a host cell, resultingin expression of the introduced sequence. In one embodiment, vectorscomprise a promoter and one or more control elements (e.g., enhancerelements) that are heterologous to the introduced DNA but are recognizedand used by the host cell. In another embodiment, the sequence that isintroduced into the vector retains its natural promoter that may berecognized and expressed by the host cell (Bormann et al., J. Bacteriol1996; 178:1216-1218).

Vectors typically comprise the DNA of a transmissible agent, into whichforeign DNA is inserted. A common way to insert one segment of DNA intoanother segment of DNA involves the use of enzymes called restrictionenzymes that cleave DNA at specific sites (specific groups ofnucleotides) called restriction sites. A “cassette” refers to a DNAcoding sequence or segment of DNA that codes for an expression productthat can be inserted into a vector at defined restriction sites. Thecassette restriction sites are designed to ensure insertion of thecassette in the proper reading frame. Generally, foreign DNA is insertedat one or more restriction sites of the vector DNA, and then is carriedby the vector into a host cell along with the transmissible vector DNA.A segment or sequence of DNA having inserted or added DNA, such as anexpression vector, can also be called a “DNA construct”. A common typeof vector is a “plasmid”, which generally is a self-contained moleculeof double-stranded DNA, usually of bacterial origin, that can readilyaccept additional (foreign) DNA and which can be readily introduced intoa suitable host cell. A plasmid vector (naked DNA) often contains codingDNA and promoter DNA and has one or more restriction sites suitable forinserting foreign DNA. Coding DNA is a DNA sequence that encodes aparticular amino acid sequence for a particular protein or enzyme.Promoter DNA is a DNA sequence which initiates, regulates, or otherwisemediates or controls the expression of the coding DNA. Promoter DNA andcoding DNA may be from the same gene or from different genes, and may befrom the same or different organisms. Recombinant cloning vectors willoften include one or more replication systems for cloning or expression,one or more markers for selection in the host, e.g. antibioticresistance, and one or more expression cassettes. Vector constructs maybe produced using conventional molecular biology and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); F. M. Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

The terms “express” and “expression.” mean allowing or causing theinformation in a gene or DNA sequence to become manifest for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g., theresulting protein, may also be said to be “expressed” by the cell.

The terms “transfection” or “transformation” means the introduction of anucleic acid into a cell, i.e. an extrinsic or extracellular gene, DNAor RNA sequence to a cell, so that the host cell will express theintroduced gene or sequence to produce a desired substance, typically aprotein or enzyme coded by the introduced gene or sequence. Theintroduced gene or sequence may also be called a “cloned” or “foreign”gene or sequence, may include regulatory or control sequences, such asstart, stop, promoter, signal, secretion, or other sequences used by acells genetic machinery. The gene or sequence may include nonfunctionalsequences or sequences with no known function. A host cell that receivesand expresses introduced DNA or RNA has been “transformed” or“transfected” and is a “transformant” or a “clone.” The DNA or RNAintroduced to a host cell can come from any source, including cells ofthe same genus or species as the host cell, or cells of a differentgenus or species.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown or used or manipulated in any way for theproduction of a substance by the cell. For example, a host cell may beone that is manipulated to express a particular gene, a DNA or RNAsequence, a protein or an enzyme. Host cells may be cultured in vitro orin vivo in one or more cells in a non-human animal (e.g., a transgenicanimal or a transiently transfected animal). For the present invention,host cells include but are not limited to Streptomyces species, E. coli,and human fibroblasts.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. In a specific embodiment, the host cell of the present inventionis a Gram-negative or Gram-positive bacteria. These bacteria include,but are not limited to, E. coli and Streptomyces species. An example ofa Streptomyces species that may be used includes, but is not limited to,Streptomyces hygroscopicus. In another embodiment, the host cell is ahuman fibroblast.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is an element operativelyassociated with a different gene than the one it is operativelyassociated with in nature.

The terms “mutant” and “mutation.” mean any detectable change in geneticmaterial, e.g. DNA, or any process, mechanism, or result of such achange. This includes gene mutations, in which the structure (e.g. DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g. protein or enzyme)expressed by a modified gene or DNA sequence.

The term “variant” may also be used to indicate a modified or alteredgene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant. Twospecific types of variants are “sequence-conservative variants”, apolynucleotide sequence where a change of one or more nucleotides in agiven codon position results in no alteration in the amino acid encodedat that position, and “function-conservative variants”, where a givenamino acid residue in a protein or enzyme has been changed withoutaltering the overall conformation and function of the polypeptide. Aminoacids with similar properties are well known in the art. Amino acidsother than those indicated as conserved may differ in a protein orenzyme so that the percent protein or amino acid sequence similaritybetween any two proteins of similar function may vary and may be, forexample, from 70% to 99% as determined according to an alignment schemesuch as by the Clustal Method, wherein similarity is based on thealgorithms available in MEGALIGN. A “function-conservative variant” alsoincludes a polypeptide or enzyme which has at least 60% amino acididentity as determined by BLAST or FASTA alignments, preferably at least75%, more preferably at least 85%, and most preferably at least 90%, andwhich has the same or substantially similar properties or functions asthe native or parent protein or enzyme to which it is compared.

As used herein, the terms “homologous” and “homology” refer to therelationship between proteins that possess a “common evolutionaryorigin,” including proteins from superfamilies (e.g., the immunoglobulinsuperfamily) and homologous proteins from different species (e.g.,myosin light chain, etc.) (Reeck et al., Cell 50:667, 1987). Suchproteins (and their encoding genes) have sequence homology, as reflectedby their sequence similarity, whether in terms of percent similarity orthe presence of specific residues or motifs at conserved positions.

Accordingly, the term “sequence similarity” refers to the degree ofidentity or correspondence between nucleic acid or amino acid sequencesof proteins that may or may not share a common evolutionary origin (seeReeck et al., supra). However, in common usage and in the instantapplication, the term “homologous,” when modified with an adverb such as“highly,” may refer to sequence similarity and may or may not relate toa common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 80%, and mostpreferably at least about 90% or 95% of the nucleotides match over thedefined length of the DNA sequences, as determined by sequencecomparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Anexample of such a sequence is an allelic or species variant of thespecific genes of the invention. Sequences that are substantiallyhomologous can be identified by comparing the sequences using standardsoftware available in sequence data banks, or in a Southernhybridization experiment under, for example, stringent conditions asdefined for that particular system.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al, supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m)(melting temperature) of 55EC, can be used, e.g., 5×SSC, 0.1% SDS, 0.25%milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS). Moderatestringency hybridization conditions correspond to a higher T_(m), e.g.,40% formamide, with 5× or 6×SCC. High stringency hybridizationconditions correspond to the highest T_(m), e.g., 50% formamide, 5× or6×SCC. SCC is a 0.1SM NaCl, 0.015M Na-citrate. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T, for hybrids of nucleic acids having those sequences. Therelative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al,supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al, supra, 11.7-11.8). A minimum length for a hybridizablenucleic acid is at least about 10 nucleotides; preferably at least about15 nucleotides; and more preferably the length is at least about 20nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55EC, and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60EC; in a more preferredembodiment, the T_(m) is 65EC. In a specific embodiment, “highstringency” refers to hybridization and/or washing conditions at 68EC in0.2×SSC, at 42EC in 50% formamide, 4×SSC, or Linder conditions thatafford levels of hybridization equivalent to those observed under eitherof these two conditions.

Suitable hybridization conditions for oligonucleotides (e.g., foroligonucleotide probes or primers) are typically somewhat different thanfor full-length nucleic acids (e.g., full-length cDNA), because of theoligonucleotides' lower melting temperature. Because the meltingtemperature of oligonucleotides will depend on the length of theoligonucleotide sequences involved, suitable hybridization temperatureswill vary depending upon the oligonucleotide molecules used. Exemplarytemperatures may be 37° C. (for 14-base oligonucleotides), 48° C. (for17-base oligonucleotides), 55° C. (for 20-base oligonucleotides) and 60°C. (for 23-base oligonucleotides). Exemplary suitable hybridizationconditions for oligonucleotides include washing in 6×SSC/0.05% sodiumpyrophosphate, or other conditions that afford equivalent levels ofhybridization.

TGF-β Gene Superfamily Proteins

TGF-β gene superfamily proteins include for example, BMP proteins,growth and differentiation factor (GDF) proteins, and transforminggrowth factor proteins (TGF). The active form of most TGF-β proteinscontain one or more conserved cysteine residues, which form one or moreinterchain disulfide bonds resulting in the formation of homodimers.Proteins from different subfamilies of the TGF-β superfamily have beenobserved to exist as heterodimers. For example, BMP-15 and GDF-9 havebeen observed. BMP-15 and GDF-9 are closely related in their primarystructures and share a nearly identical spatiotemporal expressionpattern in the oocyte during folliculogenesis in mammals (Liao et al., JBiol. Chem. 2003; 278(6):3713-9). Several other members of the TGF-βprotein family also exist as heterodimers (e.g., inhibin/activin,TGF-β1/TGF-β2) (Israel et al., Growth Factors. 1996; 13(3-4):291-300).

BMP Proteins

With the exception of BMP-1, BMP proteins are a subgroup of the TGF-βgene superfamily. All BMP proteins, except for BMP-1 form homodimers andseveral heterodimeric forms have also been described. Examples of BMPheterodimers include BMP-2/3, BMP-2/4, BMP-2/5, BMP-2/6, BMP-2/7,BMP-4/3, BMP-4/5, BMP-4/6, and BMP-4/7 (Israel et al., Growth Factors.1996; 13:291-300; Suzuki et al., Biochem Biophys Res Commun. 1997 Mar.6; 232(1):153-6; Aono et al., Biochem Biophys Res Commun. 1995 May 25;210(3):670-7). BMP proteins also form heterodimers with other TGF-βsubfamily proteins. For example, BMP-2/TGF-β1, BMP-4/TGF-β1, BMP-7/GDF-7and BMP-15/GDF-9 heterodimers have been described (Israel et al., GrowthFactors. 1996; 13:291-300; Butler S J and Dodd J, Neuron. 2003;38(3):389-401; Liao et al., J Biol. Chem. 2003; 278(6):3713-9).

BMP Fusion Gene

A BMP fusion gene comprises, sequentially, a first full-length BMP genesegment excluding the first BMP stop codon, a linker, and a second,different full-length BMP gene segment excluding the second, differentBMP start codon and the nucleotide sequence encoding the second,different BMP signal peptide.

The First BMP Gene Segment

A BMP fusion gene of the present invention includes a nucleotidesequence encoding a first BMP protein component. The first BMP fusiongene can be DNA or RNA. Nucleic acid encoding the first BMP proteincomponent can be obtained from mRNA present in human osteosarcoma celllines such as U-2 OS cells or Sa-OS cells. It is also possible to obtainnucleic acid encoding the first BMP from human cell genomic DNA. Forexample, the gene encoding the first BMP can be cloned from either acDNA or a genomic library in accordance with standard protocols. A cDNAencoding the first BMP can be obtained by isolating total mRNA from anappropriate cell line, such as U2-OS cells. See, e.g., Example 1. Doublestranded cDNAs can then be prepared from the total mRNA. Subsequently,the cDNAs can be inserted into a suitable plasmid or bacteriophagevector using any one of a number of known techniques. Genes encoding thefirst BMP can also be cloned using established polymerase chain reactiontechniques. For example, a DNA vector containing a first BMP cDNA can beused as a template in PCR reactions using oligonucleotide primersdesigned to amplify a desired region of the first BMP cDNA. In apreferred embodiment, the first BMP fusion gene encodes a BMP-2 protein.

The Linker

A linker as applied to a BMP fusion gene is a nucleotide sequence withina BMP fusion gene, which encodes an amino acid sequence and bridges anucleotide sequence encoding the first BMP protein component and thenucleotide sequence encoding the second, different BMP protein componentof a BMP fusion protein. A linker according to the present invention canbe any nucleotide sequence encoding an amino acid long enough andflexible enough to permit protein folding and not so long as tointroduce additional or erroneous folds or other secondary and/ortertiary protein structures. A preferred linker has about 60 bp(encoding about 20 amino acids). An especially preferred linker encodesthe amino acid sequence (Gly₄Ser)₄ (SEQ ID NO:5).

In an embodiment of the invention, a linker is a nucleotide sequencepresent in the human genome such that the likelihood of an immunologicalreaction upon administration of a BMP fusion gene or a BMP fusionprotein is reduced relative to administration of such a gene or proteinthat did not contain a human genomic linker.

In a particular embodiment, a nucleotide sequence encoding a (Gly₄Ser)₄(SEQ ID NO:5) linker follows, in order, the start codon of a first BMPgene and the full length of the first BMP gene, excluding the stopcodon. The linker is followed by a second, different BMP gene excludingthe start codon of the second, different BMP gene and the signal peptidenucleotide sequence of the second, different BMP (i.e., following thelinker, the fusion gene encodes the second different BMP gene after thesignal peptide up to and including the second, different BMP stopcodon).

The Second, Different BMP Gene Segment

A BMP fusion gene of the invention includes a nucleotide sequenceencoding a second, different BMP protein component. The nucleic acids ofthe second, different BMP fusion gene can be DNA or RNA. Nucleic acidencoding the second different BMP protein component can be obtained frommRNA present in human osteosarcoma cell lines such as U2-OS or Sa-OS,pancreatic adenocarcinoma, normal brain tissue or normal kidney tissue.It is also possible to obtain nucleic acid encoding the second,different BMP protein from human cell genomic DNA. For example, the geneencoding the second, different BMP can be cloned from either a cDNA or agenomic library in accordance with standard protocols. A cDNA encodingthe second, different BMP protein can be obtained by isolating total inRNA from an appropriate cell line. See Example 1. Double stranded cDNAscan then prepared from the total mRNA. Subsequently, the cDNAs can beinserted into a suitable plasmid or bacteriophage vector using any oneof a number of known techniques. Genes encoding the second, differentBMP protein can also be cloned using established polymerase chainreaction techniques. For example, a DNA vector containing the second,different BMP cDNA can be used as a template in PCR reactions usingoligonucleotide primers designed to amplify a desired region of thesecond, different BMP cDNA. In a preferred embodiment, the second,different BMP fusion gene encodes a BMP-7 protein.

It is understood that, according to the present invention, the first orsecond, different BMP gene segment can be a nucleotide sequence encodinga non-BMP, TGF-β superfamily protein.

BMP Fusion Protein

A BMP fusion protein of the present invention is more potent forinducing bone formation than a BMP homodimer of either the first orsecond, different BMP protein. A BMP fusion protein is expressed as onepolypeptide, which folds into its functional configuration. Thus, a BMPfusion protein is not a heterodimer. In accordance with the presentinvention, a BMP fusion protein comprises a first BMP protein componentand a second, different BMP protein component. A preferred BMP fusionprotein according to the present invention is a BMP-2/7 fusion protein.A preferred linker according to the invention is about 20 amino acids inlength. An especially preferred linker is (Gly₄Ser)₄ (SEQ ID NO:5).

It is understood that, according to the present invention, the first BMPprotein component or the second, different BMP protein component can bea non-BMP, TGF-β superfamily protein. A BMP fusion protein according tothe invention can be any combination of a BMP protein component and asecond, different protein component selected from among the TGF-βsuperfamily proteins wherein the first and second proteins, and anintervening linker, are expressed as one polypeptide such that eachprotein component folds into its functional conformationpost-expression.

In an aspect of the present invention, the BMP fusion protein ismodified, and the modified BMP fusion protein comprises addition,removal or substitution of at least one amino acid. These amino acidsubstitutions include, but are not necessarily limited to, amino acidsubstitutions known in the art as “conservative”. For example, it is awell-established principle of protein chemistry that certain amino acidsubstitutions, entitled “conservative amino acid substitutions,” canfrequently be made in a protein without altering either the conformationor the function of the protein. Such changes include substituting any ofisoleucine (1), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine and valine (V). Methionine (M), which isrelatively hydrophobic, can frequently be interchanged with leucine andisoleucine, and sometimes with valine. Lysine (K) and arginine (R) arefrequently interchangeable in locations in which the significant featureof the amino acid residue is its charge and the differing pK's of thesetwo amino acid residues are not significant. Still other changes can beconsidered “conservative” in particular environments.

The present invention provides methods for producing a BMP fusionprotein, wherein a host cell transfected with a BMP expression vector iscultured under conditions that provide for expression of the BMP fusionprotein. In a further aspect of the invention, a BMP fusion gene istransfected into a eukaryotic cell with a BMP expression vector, theeukaryotic cell is cultured under conditions that provide for theexpression of the BMP fusion protein, and the BMP fusion proteinproduced by the cultured eukaryotic cell is recovered. In another aspectof the invention, a BMP fusion gene is transfected into a prokaryoticcell with a BMP expression vector, the prokaryotic cell is culturedunder conditions that provide for the expression of the BMP fusionprotein, and the BMP fusion protein produced by the cultured prokaryoticcell is recovered. In preferred embodiments, the present inventionprovides methods for producing a BMP-2/7 fusion protein, wherein a hostcell transfected with a BMP-2/7 expression vector is cultured underconditions that provide for expression of the BMP-2/7 fusion protein.

Methods for Making a BMP Fusion Gene

A BMP fusion gene construct can be made by methods well known to one ofordinary skill in the art. For example, a BMP fusion gene construct canbe made using serial polymerase chain reactions. In one embodiment, aBMP-2/7 fusion gene construct can be made using serial polymerasereactions. See Example 1. Briefly, total RNA can be extracted fromcells, which express both a first BMP and a second, different BMP (e.g.,U2-OS human osteoblastic cells), and converted to cDNA using reversetranscription. PCR reactions can be performed in the standard manner,wherein a master mix containing cDNA template, primers, NTPs, buffer,MgCl and taq polymerase is prepared. A first round of polymerase chainreactions can be performed using the cDNA derived from U2-OS cells asthe template to produce first BMP+linker; and linker+second, differentBMP fragments. Primers for first BMP+linker fragment are designed toproduce a PCR product in which a KpnI site is added to the 5′ end of thefirst BMP, and the first BMP stop codon is replaced with a (Gly₄Ser)₄(SEQ ID NO:5) linker at the 3′ end of the first BMP sequence. Primersfor linker+second, different BMP can be designed to produce a PCRproduct in which the signal peptide sequence at the 5′ end of thesecond, different BMP is replaced with a (Gly₄Ser)₄ (SEQ ID NO:5) linkerand a NotI site is added at the 3′ end of the second, different BMP. Asecond round of PCR can use the first BMP+linker and linker+second,different BMP PCR products as templates. Each of these fragments is gelpurified prior to the second round of PCR. The first BMP+linker andlinker+second, different BMP fragments are fused in the second round PCRusing the 5′ first BMP (forward) primer and the 3′ second, different BMP(reverse) primer. In this manner, a BMP fusion gene is produced.

Expression Vectors, Host Cells, and Methods for Producing a BMP FusionProtein

The BMP fusion protein of the invention can be expressed byincorporating a chimeric BMP fusion gene described herein into anexpression vector and introducing the expression vector into anappropriate host cell. Accordingly, the invention further pertains toexpression vectors containing a BMP fusion gene and to host cells intowhich such expression vectors have been introduced.

An expression vector of the invention can be used to transfect cells,either prokaryotic or eukaryotic (e.g., mammalian, insect or yeastcells) to thereby produce fusion proteins encoded by nucleotidesequences of the vector. Expression in prokaryotes is most often carriedout in E. coli with vectors containing constitutive or induciblepromoters. Certain E. coli expression vectors (so called fusion-vectors)are designed to add a number of amino acid residues to the expressedrecombinant protein, usually to the amino terminus of the expressedprotein. Such fusion vectors typically serve three purposes: 1) toincrease expression of recombinant protein; 2) to increase thesolubility of the target recombinant protein; and 3) to aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. Examples of fusion expression vectors includepGEX (Amrad Corp., Melbourne, Australia) and pMAL (New England Biolabs,Beverly, Mass.) which fuse glutathione S-tranferase and maltose Ebinding protein, respectively, to the target recombinant protein.Accordingly, a BMP fusion gene may be linked to additional codingsequences in a prokaryotic fusion vector to aid in the expression,solubility or purification of the fusion protein. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the target recombinant protein toenable separation of the target recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene 69:301-315) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89). Target gene expression from the pTrc vector relies onhost RNA polymerase transcription from the hybrid trp-lac fusionpromoter. Target gene expression from the pET 11d vector relies ontranscription from the T7 gn10-lac 0 fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident.lambda. prophage harboring a T7 gn1 under the transcriptional controlof the lacUV 5 promoter.

Alternatively, a BMP fusion protein can be expressed in a eukaryotichost cell, such as mammalian cells (e.g., Chinese hamster ovary cells(CHO) or NS0 cells), insect cells (e.g., using a baculovirus vector) oryeast cells. Other suitable host cells are known to those skilled in theart. For expression in mammalian cells, the expression vector's controlfunctions are often provided by viral material. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. To express a BMP fusion protein in mammalian cells,generally COS cells (Gluzman, Y., (1981) Cell 23:175-182) are used inconjunction with such vectors as pCDM8 (Seed, B., (1987) Nature 329:840)for transient amplification/expression, while CHO (dhfr.sup.—ChineseHamster Ovary) cells are used with vectors such as pMT2PC (Kaufman etal. (1987), EMBO J. 6:187-195) for stable amplification/expression inmammalian cells. A preferred cell line for production of recombinantprotein is the NS0 myeloma cell line available from the ECACC (catalog#85110503) and described in Galfre, G. and Milstein, C. ((1981) Methodsin Enzymology 73(13):3-46; and Preparation of Monoclonal Antibodies:Strategies and Procedures, Academic Press, N.Y., N.Y.). Examples ofvectors suitable for expression of recombinant proteins in yeast (e.g.,S. cerivisae) include pYepSec1 (Baldari. et al., (1987) Embo J.6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88(Schultz et al., (1.987) Gene 54:113-123), and pYES2 (InvitrogenCorporation, San Diego, Calif.). Baculovirus vectors available forexpression of proteins in cultured insect cells (SF 9 cells) include thepAc series (Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and thepVL series (Lucklow, V. A., and Summers, M. D., (1989) Virology170:31-39).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory textbooks.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate DNA into their genomes. In order toidentify and select these integrants, a gene that encodes a selectablemarker (e.g., resistance to antibiotics) is generally introduced intothe host cells along with the gene of interest. Preferred selectablemarkers include those which confer resistance to drugs, such as G418,hygromycin and methotrexate. Nucleic acid encoding a selectable markermay be introduced into a host cell on the same plasmid as the gene ofinterest or may be introduced on a separate plasmid. Cells containingthe gene of interest can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die). The surviving cells can then be screened forproduction of BMP fusion protein by, for example, immunoprecipitationfrom cell supernatant with an anti-BMP fusion protein monoclonalantibody.

The invention also features methods for producing BMP fusion protein.For example, a host cell transfected with a nucleic acid vectordirecting expression of a BMP fusion gene can be cultured in a mediumunder appropriate conditions to allow expression of the protein tooccur. Suitable mediums for cell culture are well known in the art.Protein can be isolated from cell culture medium, host cells, or bothusing techniques known in the art for purifying proteins. In a oneembodiment, the invention features methods for producing BMP-2/7 fusionprotein.

Methods for Treatment

A BMP fusion protein or BMP fusion gene of the invention can beincorporated into compositions suitable for administration to patientsto induce bone formation for the treatment of bone loss. Administrationof a BMP fusion protein or BMP fusion gene as described herein can be inany pharmacological form including a therapeutically effective amount ofBMP fusion protein or BMP fusion gene and a pharmaceutically acceptablecarrier. In an aspect of the present invention, a BMP-2/7 fusion proteinor a BMP-2/7 fusion gene can be incorporated into compositions suitablefor administration to patients to induce bone formation for thetreatment of bone loss.

The active compound (e.g., BMP fusion protein or BMP fusion gene) can beadministered in a convenient manner such as by topical application(i.e., direct application to the bone or to the vicinity of the bone),injection (intra-osseous, intra-articular, subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, theactive compound may be coated in a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound. Preferred routes of administration are topical(i.e., direct application to the bone or to the vicinity of the bone),percutaneous (e.g., intra-osseous or intra-articular injection), andintravenous. Systemic (e.g., intravenous) BMP fusion gene therapy may betargeted to the site of bone loss as by, for example, targetingosteoblasts using an osteoblast-specific promoter. For example, a viralvector containing a BMP-2/7 fusion gene can be modified such that geneexpression is regulated and replication of the viral vector isrestricted to cells capable of activating an osteocalcin promoter (Kuboet al., Hum Gene Ther. 2003; 14(3):227-41; Hsieh et al., Cancer Res.2002; 62(11):3084-92).

In one embodiment, a BMP fusion protein such as BMP-2/7 fusion proteinor BMP-2/7 fusion gene such as BMP-217 is administered topically (i.e.,direct application to the bone or to the vicinity of the bone). Thetopical route of administration is advantageous because the site of boneloss requiring treatment is frequently exposed as a result of surgery ortrauma and, thus, the BMP fusion protein or Bmp fusion gene can bedirectly applied to the target site. Pharmaceutically acceptablecarriers especially suited for topical administration (i.e., directapplication to the bone or to the vicinity of the bone) include gelatinhemostasis sponge (Gelfoam), Type I collagen gel, deactivateddemineralized bone matrix, and any carrier used for the topical deliveryof rhBMP2 or 7. For example, an area of bone loss can be associated withbleeding from cut bone edges. A BMP fusion protein in the form of apaste or gel can be applied to a gelatin hemostasis sponge, which isapplied to the area of bleeding and bone loss. In this manner, thepatient can be treated for both bone loss and the bleeding. In oneembodiment of the invention, the BMP fusion protein or BMP fusion geneis delivered directly to the site of bone loss in combination with amatrix providing a structure for developing bone. Matrix material caninclude, for example, calcium sulfate, tricalciumphosphate,hydroxapatite, polylactic acid, polyglycolic acid, polyanhydrides, andmixtures thereof. According to the present invention, BMP fusion proteinor BMP fusion gene can be co-administered with autograft or allograftbone.

Generally, topical administration (i.e., direct application to the boneor to the vicinity of the bone) of a BMP fusion gene or BMP fusionprotein will be a one-time application. Typically, the one-time topicaladministration will be applied prior to closure of a traumatic wound orother defect that has resulted in exposed bone (e.g., a patient with anopen bone fracture is administered a BMP fusion protein directly to thefracture site in an emergency room), or at the time of surgery. In somecircumstances, repeat topical dosing can be administered such as, forexample, during re-operation for non-healing bone.

A BMP fusion protein or BMP fusion gene can be in the form of a powder.A BMP fusion protein powder can be produced, for example, from thesupernatant of a producer cell line (mammalian or bacterial) geneticallymodified to overexpress BMP fusion protein, which is purified andlyophilized. In one embodiment, the BMP fusion protein powder can bewetted to form a gel or paste for topical administration (e.g.,placement into the bone gap of a fracture).

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. In all cases, the composition must be sterile and must befluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the BMPfusion protein or BMP fusion gene in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,preferred methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the active ingredient (e.g., peptide) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

A vector according to the present invention can be delivered in vivo orex vivo. In vivo delivery of a BMP fusion gene vector means that the BMPfusion gene and a pharmaceutically acceptable carrier are administereddirectly to the patient. Ex vivo delivery of a BMP fusion gene meansthat cells from the patient are transfected with the BMP vector in vitroand then the transfected cells are administered to the patient. Suitablecells for ex vivo delivery include, for example, primary marrow stromalcells, muscle stem cells, bone marrow stem cells, chondrocytes, dermalfibroblasts, and gingival fibroblasts.

Any technology suitable for delivery of a therapeutic gene, whether inthe form of naked (plasmid) DNA or other vector, is applicable to theBMP fusion gene of the present invention. A liquid suspension dosageform is especially preferred for plasmid or other vector BMP fusion genetherapy.

Administration of a therapeutically active amount of the therapeuticcompositions of the invention is defined as an amount effective, atdosages and for periods of time necessary to achieve the desired result.For example, a therapeutically active amount of a BMP fusion protein orBMP fusion gene may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of protein toelicit a desired response in the individual. Dosage regimen may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. Typically, the effects of a BMP fusion gene or BMP fusionprotein would be expected to last for at least 4 weeks. Thus, in apreferred embodiment, a dose of a BMP fusion protein or BMP fusion geneis administered about every four weeks until adequate bone has formed.

To administer a BMP fusion protein or BMP fusion gene by other thanparenteral administration, it may be necessary to coat the protein with,or co-administer the protein with, a material to prevent itsinactivation. For example, a BMP fusion protein or BMP fusion gene maybe administered to an individual in an appropriate carrier, diluent oradjuvant, co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. When the BMP fusion protein or BMPfusion gene is suitably protected, as described above, the protein maybe orally administered, for example, with an inert diluent or anassimilable edible carrier.

It is especially advantageous to formulate parenteral and topicalcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutically acceptable carrier.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingthe invention in spirit or in scope.

Example 1 Construction of cDNA Encoding a BMP2/7 Fusion Protein UsingSerial Polymerase Chain Reactions

The 5′ end of a gene encoding a BMP-2/7 fusion protein begins with thestart codon of BMP2 and continues through the full length of the BMP2gene, excluding the stop codon. The BMP2 nucleotide sequence is followedby a (Gly₄Ser)₄ (SEQ ID NO:5) linker, which replaces the stop codon ofthe BMP2 gene, the start codon of the BMP7 gene, and the signal peptidenucleotide sequence of BMP7. Following the linker, the fusion geneencodes the remainder of the BMP7 gene (i.e., the BMP7 gene after thesignal peptide) up to and including the BMP7 stop codon. See FIG. 1.

Using Trizol reagent (Sigma) according to the manufacturer'sinstructions, total RNA was extracted from U2-OS human osteoblasticcells, which express both BMP2 and BMP7. Reverse transcription wasperformed to convert the RNA to cDNA.

PCR reactions were performed in a standard, well known manner. Briefly,a master mix containing cDNA template, primers, NTPs, buffer, MgCl andtaq polymerase was prepared. The PCR reactions were run according to thefollowing protocol:

Thermo-cycling: 94 degrees C. for 3 minutes;

35 cycles of:

-   -   94 degrees C. for 30 seconds,    -   55 degrees C. for 1 minute, and    -   68 degrees C. for 2 minutes;

68 degrees C. for 5 minutes.

The first round of polymerase chain reactions (PCRs) was performed usingcDNA derived from U2-OS cells as the template to produce BMP2+linker,and linker+BMP7 fragments.

Primers for BMP2+linker were designed to produce a PCR product in whicha KpnI site was added to the 5′ end of BMP2, and the BMP2 stop codon wasreplaced with the (Gly₄Ser)₄ (SEQ ID NO:5) linker at the 3′ end of theBMP2 sequence. Forward primer: (SEQ ID NO: 6)GGTACCACCATGGTGGCCGGGACCCGCTGTCTT Reverse primer: (SEQ ID NO: 7)ACTTCCACCTCCACCACTACCACCTCCTCCACTACCTCCACCTCCACTTCCTCCACCACCGCGACACCCACAACCCTCCACAAC

The expected PCR product BMP2+linker=1.19 kb+60 bp=1.25 kb.

Primers for linker+BMP7 were designed to produce a PCR product in whichthe signal peptide sequence at the 5′ end of BMP7 was replaced with the(Gly₄Ser)₄ (SEQ ID NO:5) linker and a NotI site was added at the 3′ endof BMP7. Their sequences are: Forward primer: (SEQ ID NO: 8)GGTGGTGGAGGAAGTGGAGGTGGAGGTAGTGGAGGAGGTGGTAGTGGTGGAGGTGGAAGTGACTTCAGCCTGGACAACGAGGTG Reverse primer: (SEQ ID NO: 9)GCGGCCGCCTAGTGGCAGCCACAGGCCCGGAC

The expected PCR product linker+BMP7=60 bp+1.317 kb=1.377 kb The secondround of PCR used the BMP2+linker and linker+BMP7 PCR products astemplates. Each of these fragments was gel purified prior to the secondround of PCR. The BMP2+linker and linker+BMP7 fragments were fused inthe second round PCR using the 5′ BMP2 (forward) primer and the 3′ BMP7(reverse) primer. Their sequences are: Forward primer: (SEQ ID NO: 6)GGTACCACCATGGTGGCCGGGACCCGCTGTCTT Reverse primer: (SEQ ID NO: 9)GCGGCCGCCTAGTGGCAGCCACAGGCCCGGAC

The expected PCR product (i.e., BMP2+linker+BMP7) was 2.567 Kb.

Example 2 The BMP-2/7 Fusion Gene has been Cloned into an ExpressionVector and Transfected into a Producer Cell Line

The BMP2+linker+BMP7 PCR product was gel purified and cloned into TopoPCR 2.1 vector (available from Invitrogen). The cDNA sequence wasconfirmed. The BMP2+linker+BMP7 PCR product cloned into Topo PCR 2.1vector was subcloned into the pShuttleCMV (available from Stratagene)expression vector. The BMP-2/7 fusion gene expression vector wastransfected into a producer cell line (human lung epithelial carcinomaA549 cells). The supernatant of the transfected cells was shown tocontain BMP-2/7 fusion protein by immunoprecipitation.

To assess the production of BMP-2/7 fusion protein, pShuttleCMV-BMP-2/7plasmid DNA was used to transfect A549 cells via a polyfection method(QIAGEN). As controls, A549 cells were transfected with a plasmidencoding the marker gene green fluorescent protein (pCMV-GFP) or noplasmid (“medium”). Forty-eight hours after transfection, supernatantswere harvested. To confirm the presence of BMP-2/7 fusion protein, cellsupernatants were immunoprecipitated with anti-BMP2 antibody (antibodiesavailable from R&D Systems, Minneapolis, Minn.; Seize Ximmunoprecipitation kit available from Pierce Biotechnology, Rockford,Ill.) and then by BMP7 ELISA (antibodies available from R&D Systems).Only supernatant of cells transfected with pShuttleCMV-BMP-2/7, whichhad been immunoprecipitated with anti-BMP2 antibody, contained BMP7.This indicated the presence of BMP-2/7 fusion protein in this group. SeeFIG. 4. Controls did not contain BMP-2/7 fusion protein. Similar resultswere observed When cell supernatants were immunoprecipitated withanti-BMP7 antibody and followed by BMP2 ELISA.

These experiments showed that BMP-2/7 fusion gene cloned into anexpression vector and transfected into a producer cell line producedBMP-2/7 fusion protein.

Example 3 The BMP-2/7 Producer Cell Supernatant was Equipotent toBMP-2/7 Heterodimer Produced by Co-Transfection

BMP stimulation in vitro prevents mouse myoblast C2C12 cells fromdeveloping into muscle cells and induces these cells to differentiateinto bone-type cells (osteoblasts). C2C12 expression of osteocalcin(OCN), a protein important for matrix mineralization by osteoblasts, wasused as a measure of osteoblast differentiation.

C2C12 cells were stimulated for 7 days with supernatants of A549producer cells transfected with pShuttleCMV-BMP2/7 plasmid DNA orco-transfected with adenovirus vector encoding BMP2 (AdBMP2) and anotheradenovirus vector encoding BMP7 (AdBMP7). As controls, A549 cells weretransfected with a plasmid encoding the marker gene green fluorescentprotein (pCMV-GFP) or no plasmid (“medium”). Supernatants ofpShuttleCMV-BMP-2/7 transfected cells containing 5 ng/ml of BMP-2/7fusion protein (at 1:1 dilution) induced about 6 ng/ml of osteocalcin(OCN) expression. See FIG. 5 a. Supernatants generated fromco-transfection by BMP2 and BMP7 genes containing 4 ng/ml BMPs (1:80dilution) or 8 ng/ml BMPs (1:40 dilution) induced about 2 ng/ml and 8ng/ml of OCN expression in C2C12 cells, respectively. See FIG. 5 b.

These experiments showed that BMP-2/7 fusion protein is equipotent toBMP-2/7 heterodimer.

Example 4 In Vitro Dose Response Studies Compared BMP-2/7 Heterodimerwith rhBMP2 and rhBMP7

OCN concentration was used to measure osteoblast differentiation inC2C12 cells. BMP-2/7 fusion protein administered at a concentration of 2ng/ml resulted in an OCN level comparable to the OCN level that resultedfrom administration of 1000 ng/ml of rhBMP2 or rhBMP7. See FIG. 5 a.

Dose response studies using this C2C12 system also showed that a maximumresponse to BMP2/7 heterodimer produced by co-transfection of BMP2 andBMP7 genes occurred at a concentration of about 150 ng/ml. See FIG. 5 b.This result demonstrated that BMP-2/7 heterodimer induces OCN levelsthat are about 6-fold higher than can be induced by maximal doses (i.e.,1000 ng/ml) of rhBMP2 or rhBMP7. See FIG. 6.

These results confirmed that BMP-2/7 heterodimer is a more potentinducer of osteoblast differentiation than rhBMP2 or rhBMP7. Thus, aBMP-2/7 fusion protein equipotent to a BMP-2/7 heterodimer should be amore potent inducer of osteoblast differentiation than rhBMP2 or rhBMP7.

Example 5 Reverse Transcriptase-PCR (RT-PCR) of Cells Transfected withpSCMV-BMP 2/7 Plasmid

A549 epithelial cells were transfected with pCMV-BMP2/7, PCMV-GFP, ormedium-only control. Two days later RT-PCR was performed. FIG. 35. A2.56 kb band, the expected size for a BMP 2/7 fusion gene, was detectedin the cells transfected with pSCMV-BMP 2/7 (Lane 2), but not in cellstransfected with pCMV-GFP (Lane 3) or medium-only control (Lane 4).Neither BMP 2 cDNA transcripts alone (expected size 1.2 kb) nor BMP 7cDNA transcripts alone (expected size 1.4 kb) were detected inpSCMV-BMP2/7 transfected cells. Moreover, BMP2/7 fusion gene was notamplified from RNA of cells transfected with pSCMV-BMP2/7 without theaddition of reverse transcriptase. Thus, the amplified BMP2/7 fusiongene product was not contributed directly by plasmid DNA ofpSCMV-BMP2/7.

These experiments showed that pSCMV-BMP2/7 transfection resulted in theproduction of only BMP2/7 fusion gene, and not BMP2 gene alone or BMP7gene alone.

Example 6 The Supernatant of pSCMV-BMP2/7 Transfected Cells ContainBMP2/7 Fusion Protein

Western blotting was performed on the supernatant of A549 epithelialcells transfected with pSCMV-BMP2/7 using anti-BMP2 antibody. FIG. 36The majority of mature BMP peptides in the supernatant migrated as animmunoreactive band at approximately 39 kDa under non-reducingconditions. This is the expected size for a peptide composed of BMP2,linker, and BMP7 (Lane 1). B-mecacaptoethanol was added and, underreducing conditions, some mature peptides migrated further as productswith molecular masses between approximately 15 to approximately 18 kDa(Lane 1A). This suggested that the 39 kDa fusion gene product wasseparated into monomers. A similar pattern of migration of mature BMPpeptides (at approximately 39 kDa, and between approximately 15 kDa toapproximately 18 kDa) was detected by anti-BMP2 antibody in supernatantsfrom cells transfected with pSCMV-BMP2/7 that had beenimmunoprecipitated with either anti-BMP7 antibody (Lane 4) or anti-BMP2antibody (Lane 6) prior to Western blotting. The “flow-through” portionof the samples collected from the immunoprecipitation columns did notshow bands reacting with anti-BMP-2 antibody (Lanes 7-10). A broad bandbetween 45 kDa to 55 kDa was detected in cells transfected withpSCMV-BMP2/7 (Lanes 4 and 6). This finding indicated the presence ofpro-forms of BMP2/7 protein and was consistent with previous studieswhich found that BMP2 and BMP7 are processed as pro-peptides duringprotein synthesis. Western blotting using anti-BMP7 antibody yieldedsimilar results (data not shown), which indicated that BMP2/7 fusionprotein, but not BMP7 homodimer, had been produced by the BMP2/7 fusiongene-transfected cells.

The levels of BMP2 and BMP7 measured by ELISA were similar in thesupernatants of cells transfected with pSCMV-BMP2/7. FIG. 37 Controlsupernatants did not contain detectable BMP levels. Afterimmunoprecipitation with anti-BMP2 antibody, the supernatants of cellstransfected with pSCMV-BMP2/7 contained BMP7 as measured by ELISA. FIG.38 Controls did not contain detectable BMP7.

1. An BMP fusion gene.
 2. The BMP fusion gene according to claim 1,wherein the BMP fusion gene is a BMP-2 fusion gene.
 3. The BMP fissiongene according to claim 2, wherein the BMP fusion gene is a humanBMP-2/7 fusion gene.
 4. The BMP fission gene of claim 1, which comprisesa nucleotide sequence that encodes a first BMP protein, a linker, and anucleotide sequence that encodes a second, different BMP protein.
 5. TheBMP-2/7 fusion gene according to claim 4, wherein the nucleotidesequence that encodes a first BMP protein encodes BMP-2 protein, alinker, and the nucleotide sequence that encodes a second, differentencodes BMP-7 protein.
 6. The BMP fission gene according to claim 4,wherein the linker is a nucleotide sequence having about 60 base pairs.7. The BMP fusion gene according to claim 6, wherein the linker encodesan amino acid having a sequence (Gly₄Ser)₄ (SEQ ID NO:5).
 8. Apharmaceutical composition, which comprises the BMP fusion gene of claim2 and a pharmaceutically acceptable carrier.
 9. A BMP fusion protein.10. The BMP fusion protein according to claim 9, wherein the BMP fusionprotein is a BMP-2/7 fusion protein.
 11. The BMP fusion proteinaccording to claim 10, wherein the BMP fusion protein is a human BMP-2/7fusion protein.
 12. The human BMP fusion protein of claim 11, whereinthe BMP fusion protein comprises: (a) a human first BMP protein aminoacid sequence; (b) a linker; and (c) a human second, different BMPprotein amino acid sequence.
 13. The human BMP fusion protein of claim12 wherein the linker is (Gly₄Ser)₄ (SEQ ID NO:5).
 14. The BMP fusionprotein according to claim 9, which comprises: (a) a first BMP aminoacid sequence as set forth in any one of SEQ ID NOS:2, 4 or 10 to 64;(b) a linker as set forth in SEQ ID NO:5; and (c) a second, differentBMP amino acid sequence as set forth in any one of any one of SEQ IDNOS:2, 4, or 10 to 64; wherein the BMP amino acid sequence of (a) isdifferent than the BMP amino acid sequence of (b) and either (a) or (b)is a BMP amino acid sequence as set forth in any one of SEQ ID NOS:2, 4or 10 to
 39. 15. The human BMP-2/7 fusion protein of claim 10, whereinthe fusion protein comprises: (a) a human BMP2 amino acid sequence asset forth in SEQ ID NO:2; (b) a linker as set forth in SEQ ID NO:5; and(c) a human BMP7 amino acid sequence as set forth in SEQ ID NO:4;wherein the linker replaces the BMP2 stop codon, the BMP7 start codon,and the BMP7 signal peptide nucleotide sequence.
 16. A BMP fusion genecomprising an isolated nucleic acid having a nucleotide sequenceselected from the group consisting of: (a) a nucleic acid sequence thathybridizes to a nucleotide sequence encoding a fusion protein as setforth in claim 10, said hybridization being performed under stringentconditions; (b) a nucleic acid sequence encoding a polypeptide at least90% homologous to a fusion protein as set forth in claims 10; and (c) anisolated nucleic acid fragment having a nucleotide sequencecomplementary to the nucleotide sequence of (a) or (b).
 17. Apharmaceutical composition comprising a BMP fusion protein according toclaim 10 and a pharmaceutically acceptable carrier.
 18. An expressionvector which comprises the BMP fusion gene of claim 4 operativelyassociated with an expression control sequence.
 19. A method forproducing a BMP fusion protein which comprises: (1) fusing a linker tothe 3′ end of a first BMP gene; and (2) fusing a second, different BMPgene to the 3′ end of the linker; wherein the linker replaces the firstBMP gene stop codon; the second, different BMP start codon; and thesecond, different BMP signal peptide nucleotide sequence.
 20. The methodaccording to claim 19, wherein the first BMP gene is a BMP-2 gene andthe second, different BMP gene is a BMP-7 gene.
 21. A method forinducing bone growth in a patient, which comprises administering a BMPfusion protein to a patient in need thereof.
 22. The method according toclaim 21, wherein the BMP fusion protein is administered directly to abone.
 23. The method according to claim 21, wherein the BMP fusionprotein is administered in the vicinity of a bone.
 24. A method forinducing bone growth in a patient, which comprises administering a BMPfusion gene to a patient in need thereof.
 25. A host cell comprising theexpression vector of claim
 18. 26. A method for producing a BMP-2/7fusion protein, which method comprises isolating the BMP-2/7 fusionprotein produced by the host cell of claim 25, wherein the host cell hasbeen cultured under conditions that provide for expression of theBMP-2/7 fusion protein.
 27. A BMP fusion gene according to claim 1,further comprising a nucleotide sequence encoding a BMP proteincomponent, a linker, and a nucleotide sequence encoding a TGF-βsuperfamily protein component, wherein the TGF-β superfamily proteincomponent is different than the BMP protein component.
 28. The BMPfusion gene according to claim 27, wherein the BMP fusion gene encodes aBMP fusion protein selected from the group consisting of: BMP-7/GDF-7;BMP-15/GDF-9; BMP-2/TGF-β1 and BMP-4/TGF-β1.
 29. A BMP fusion proteinaccording to claim 9, further comprising a BMP protein component, alinker, and a nucleotide sequence encoding a TGF-β superfamily proteincomponent, wherein the TGF-β superfamily protein component is differentthan the BMP protein component.
 30. The BMP fusion protein according toclaim 29, wherein the BMP fusion protein is selected from the groupconsisting of: BMP-7/GDF-7; BMP-15/GDF-9; BMP-2/TGF-β1 and BMP4/TGF-β1.